Barley rhizobacterial population characterised by fatty acid pro®ling
Stig Olsson
*, Sadhna AlstroÈm, Paula Persson
Department of Ecology and Crop Production Science, Swedish University of Agricultural Sciences, Box 7044, Uppsala S-75007, Sweden
Received 27 October 1998; received in revised form 9 February 1999; accepted 12 February 1999
Abstract
The aim of the study was to explore the possibilities for using fatty acid analysis to characterise rhizobacterial populations. Fatty acid pro®les were determined for 1188 bacterial isolates originating from barley roots growing in three different soils, two of which were clays and one a silt loam. A multivariate statistical treatment of the fatty acid data revealed three distinct groups of rhizobacteria. For 720 isolates (Group A), almost the whole fatty acid content of the cell consisted of straight-chain fatty acids with an even number of carbon atoms in the chains. In another group (B) of 435 isolates, most of the fatty acids had branched chains with an odd number of carbon atoms. A small group (C) with 33 isolates was mainly characterised by high contents of the fatty acid 15 : 0 Anteiso. The dominating bacterial genera in these three groups werePseudomonas,Cytophaga
and Gram positives, respectively. When roots had grown in clay soils bacteria from Group A were most frequent, Group B dominated in the loam.#1999 Elsevier Science B.V. All rights reserved.
Keywords:Rhizobacteria; FAME;Cytophaga;Flavobacterium;Pseudomonas
1. Introduction
Recognition that the rhizobacterial population affects plant growth, has led to the initiation of a great number of investigations, aimed at better understand-ing and explorunderstand-ing the potential bene®ts of these bacteria in agricultural production. In these studies soil-borne and/or root-colonising bacteria have been screened for their promoting or deleterious effects on plant growth and for antagonistic properties against root diseases (Nehl et al., 1996).
In some studies the composition of the rhizobacter-ial population has been described by characterising the chemical composition and/or metabolic activity of individual isolates. Kloepper et al. (1992); Lilley
et al. (1996) used the composition of whole cell fatty acids, and Lambert et al. (1990) used cellular proteins to group and identify their isolates. Sato and Jiang (1996a, b) combined a DNA analysis with other chemical tests to group bacteria sampled from wheat roots. Colony development (de Leij et al., 1993) and metabolic potentials (Kleeberger et al., 1983) have also been used together with standard tests for identi-fying bacteria.
The common approach taken in the above studies has been to describe the composition of a population by characterising some of its members. This approach introduces some methodological problems that make it dif®cult to interpret the results. First, they only include the cultivable bacteria, which account for only a small fraction of the total bacterial population (Olsen and Bakken, 1987). This is not, however, a serious drawback in studies aimed atcomparingpopulations
*Corresponding author. Fax: +46-18-672890; e-mail: stig.olsson@vpat.slu.se
from different soils or habitats. Moreover, when study-ing rhizobacteria and usstudy-ing appropriate incubation conditions, the cultivable fractions are most likely to be the ones with the highest in situ metabolic activity (Olsen and Bakken, 1987).
Another drawback in their approach is related to the dif®culty in sampling representative sets of cultivable bacteria from heterogeneous media such as soil, rhi-zosoil and roots. In some of the above mentioned studies, the general validity of the results is restricted due to the fact that it has not been possible to handle a suf®ciently large number of individual isolates. This problem could partly be solved, if analysis and char-acterisation of individual isolates could be combined with the analysis of the whole population by the same set of methods. A combined approach could charac-terise a larger number of populations and, thus, it would be possible to reach more general conclusions. The analysis of the whole cell fatty acid content could be used for such investigations.
Fatty acid analysis is a well-established method for identifying bacterial strains (Sasser, 1990). It is a fast and cheap method, and a large number of conservative characteristics can be subjected to quantitative analy-sis. The fatty acid contents could be extracted from the in situ populations in soil or from plated populations (Cavigelli et al., 1995; Zelles et al., 1995). The method can be used for describing individual bacterial isolates as well as mixed bacterial populations (Haack et al., 1994). It is likely that the fatty acid pro®les of mixed populations will re¯ect the pro®les of the bacteria they are composed of.
The aim of the present study was to explore the possibilities for using fatty acid pro®ling to character-ise bacterial populations. In this paper we take the ®rst step and describe the fatty acid pro®les of separate isolates obtained from rhizobacterial populations on barley roots. In a second paper Olsson and Persson (1999) we explore these results to analyse the fatty acid pro®les of mixed bacterial populations from different habitats in soil.
2. Materials and methods
2.1. Soils and soil samples
Soil was sampled in September 1995 and May 1996 from three experimental ®elds with long-term crop
rotations. The ®elds were located at Ultuna, LoÈvsta and SaÈby, situated near Uppsala and managed by the Department of Crop Production Science, Swedish University of Agricultural Sciences. At Ultuna and LoÈvsta the soils were heavy clays and at SaÈby a silt loam. Further physico-chemical properties of the soils were described by Olsson and Gerhardsson (1992). Soils were taken from two rotational treatments; one in which barley had been cultivated in rotation with other crops and another in which barley had been the only crop planted for the last 25 years.
Soil samples (ca 500 g fresh weight) were taken with an auger from the upper 15 cm soil layer. Each sample consisted of 8±10 mixed sub-samples taken from a small square (approximately 2020 cm) in the ®eld. Three such samples were taken in each replicated plot. The soils were sieved (mesh size 4 mm) to create suitable media for cultivating the bait crop, barley (cv. `Golf'), from the roots of which the bacterial populations were to be sampled. Whenever the soil moisture content was too high to allow the samples to be sieved directly, they were dried down to a level where they could handled (approxi-mately 15% water content).
The soil sampled in September 1995 was used to prepare soil-testing strips for use in specially designed plant-cultivating equipment. In this equipment the barley bait plants are grown for 14 days with their roots in intimate contact with a thin layer of soil sandwiched between a strip of greenhouse watering web and a thin transparent net. Such a web-soil-net arrangement is called a soil testing strip. The strips were inserted into Plexiglas tubes with a recirculating plant nutrient solution. This equipment is described in more details by Olsson and Kadir (1994); Olsson and AlstroÈm (1996).
The soil sampled in May 1996 was lightly packed into tube-shaped, polyethylene plastic bags (100 cm long and 3.2 cm in diameter) which were used as growing pots. Five pregerminated seeds of barley were planted in each tube, which was placed in a climate chamber with 12 h light and a constant temperature of 118C. After 17±21 days the barley roots were har-vested.
2.2. Bacterial sampling
adhering soil particles were removed, and the roots were macerated with a glass tissue grinder in 5 ml sterile 0.01 M MgSO4 solution. This root-rhizosoil-microbiota suspension was further diluted, plated on TSA medium (15 g lÿ1
trypticase soybroth10 g lÿ1 Oxoid technical agar) and incubated for 4 or 5 days at 118C. Approximately 15 colonies were sampled from each plate. In addition, an attempt was made to obtain a sample re¯ecting the composition of the population on the plate in terms of visually characterisable prop-erties, e.g. size, colour, colony shape. In this way bacterial isolates were collected from 232 23 (occasions®eldscrop rotations repli-cated plots per rotation and ®eldreplicated samples per plot), making a total of 72 different root samples.
2.3. Fatty acid extraction and analysis
For fatty acid analysis of the sampled bacteria, the bacterial strains were grown on TSA (20 g lÿ1
TSB10 Oxoid g lÿ1
technical agar) and incubated for 24 h at 248C rather than according to the MIS standard procedure (30 g TSB and 288C). The modi®cations were motivated by the results of pre-liminary tests in which many isolates sampled from roots grew poorly under the recommended standard conditions. To control the effects of the introduced modi®cations on incubation conditions, 14 isolates were analysed after being incubated according to both routines. Approximately 50 mg fresh weight of cells was harvested, and the fatty acid methyl esters (FAMEs) were extracted as described by Sasser (1990)
FAMEs were separated on a Hewlett Packard 5890 Series II gas chromatograph with a 25 m0.2 mm methyl silicone fused silica capillary column, using hydrogen as carrier gas. Individual FAMEs were identi®ed and quanti®ed using the peak-naming table component of the microbial identi®cation system (MIS, Microbial ID, US). Their relative quantities were expressed as percentages of the total named FAME peak area.
2.4. Statistical analysis
A macro-routine was constructed with a spread-sheet computer program in order to transfer the MIS output format into a format read by the Systat 5.21
statistical programme, which was used for all of the statistical analyses.
The variables in the statistical analysis were the relative quantities of named fatty acids in each single isolate. The analysis of the results was performed in four consecutive steps:
(a) the fatty acids were ranked according to their amount in the bacterial population,
(b) the variation between isolates in their fatty acid composition was summarised by a principle component analysis of the dominating fatty acids, (c) the principle components were used to cluster the isolates,
(d) the characteristics of the main clusters were investigated.
3. Results
3.1. Grouping of bacterial isolates
In total, 1188 bacterial isolates were collected from 72 separate root samples. All isolates were analysed to determine their whole cell fatty acid composition. Sixty-nine distinct FAME peaks were found, of which 61 were identi®ed by the system. When comparing these fatty acids in terms of their relative amounts and frequencies in the bacterial population, great differ-ences were found between them. The 20 most abun-dant fatty acids accounted for more than 80% of the total FAME content in 95% of the isolates analysed, whereas the remaining 49 fatty acids were only found in a small fraction of the total population, in which they accounted for a minute proportion of the total amount of FAME.
The ®rst two principal components of the 20 most abundant fatty acids represented 60 and 10% respec-tively, of the total variation between isolates. The correlations between the original variables and the two components are shown in Table 1. From this table it is clear that the main factor dividing the bacterial population was the number (even versus odd) of carbon atoms in the fatty acid chains.
between the two main groups became evident. These three groups can be more strictly de®ned in the following way:
Group A1st component < 0
Group B1st component > 0 and 2nd component >ÿ1.5
Group C2nd component <ÿ1.5
When examining some alternative de®nitions, it was found that only few isolates changed group af®liation. If for example Group C was de®ned as 2nd component <ÿ1.2, six isolates changed from B to C, and if Group C2nd component <ÿ1.8, three isolates changed from C to B. Thus, it was concluded that the clustering of the bacterial populations into three main groups is robust, with very few intermedi-ate cases.
3.2. Characterisation of the main bacterial groups
Differences in fatty acid composition between the three groups are shown in Table 2. In Group A,
bacteria had about 90% of their whole cell fatty acid content as even-numbered fatty acids, whereas in Group B bacteria had mainly (approximately 70%) odd numbered fatty acids. The small Group C is characterised by its high content of 15 : 0 Anteiso.
These three main bacterial groups also correspond to different bacterial genera as identi®ed in MIS with a similarity index (SI) greater than 0.6. Group A is dominated by Pseudomonas spp. , Group B by
Cytophaga spp. and Group C by Gram positives, as shown in Table 3. The same table also shows that approximately 40% of the isolates were left unidenti-®ed; i.e. their similarity indexes (SI) were lower than 0.6.
Table 4 shows the fatty acid compositions of sub-groups within the three main Groups A, B and C. The subgroups were de®ned so as to allow the dom-inating genera of a main group to be directly compared with the other isolates of that group. Thus, for exam-ple, Group A is divided into the subgroups `Pseudo-monas' (366 isolates classi®ed by MIS with SI > 0.6) and `Not Pseudo.' (354 isolates representing minor genera and unidenti®ed isolates). As shown in Table 4, there is good agreement in the fatty acid patterns between the various subgroups within the same main group.
Table 1
Correlations between the 20 dominating fatty acids and the first two principal components identified by principal component analysis
Fatty acid 1st principal component
2nd principal component
16:0 ÿ0.92 ÿ0.02
16 : 1 / 15 : 0 Iso 2OH ÿ0.74 ÿ0.09
18 : 1 ÿ0.67 ÿ0.03
12 : 0 ÿ0.63 ÿ0.16
17 : 0 CYCLO ÿ0.54 ÿ0.01
10 : 0 3OH ÿ0.73 0.61
12 : 0 3OH ÿ0.77 0.56
12 : 0 2OH ÿ0.78 0.29
12 : 1 3OH ÿ0.33 0.80
15 : 0 Iso 0.92 ÿ0.05
17 : 1 Iso 0.82 ÿ0.01
17 : 0 Iso 3OH 0.86 0.02
15 : 0 0.85 0.21
15 : 1 0.86 0.25
17 : 1 0.87 0.26
17 : 1 Iso/Anteiso 0.86 0.19
15 : 1 Iso G 0.79 0.20
16 : 0 Iso 3OH 0.92 0.19
15 : 0 Iso 3OH 0.95 0.16
15 : 0 Anteiso 0.20 ÿ0.38
3.3. Effect of different incubation conditions
Fourteen isolates had been identi®ed asPseudomonas
spp. (seven isolates), asCytophagasp. (six isolates) and anArthrobactersp., when cultured under the modi®ed incubation conditions (20 TSB at 248C). When using the recommended MIS standard procedure (30 g TSB and incubated at 288C), the taxonomic determinations were not affected at the genus level. At the species level, however, an isolate identi®ed as P. putida under the standard conditions was identi®ed asP. marginalisunder the modi®ed conditions, and one identi®ed asP. chloro-raphisunder the standard conditions was determined to beP. putidaunder the modi®ed ones. Furthermore, an isolate identi®ed asP. chlororaphisunder the modi®ed conditions had a SI of 0.5 under the standard ones and thus was regarded as unidenti®ed.
3.4. Differences between soils
The three groups of bacteria differed in their relative abundance between the experimental ®elds as shown
in Table 5. Group A isolates were more frequent than B or C isolates in the clay soils of LoÈvsta and Ultuna, whereas Group B was the most frequent group at SaÈby where the soil is a silt loam (Table 5;p-value < 10ÿ3 in Kruskal±Wallis test under the hypothesis that the A and B groups of bacteria were equally frequent in all ®elds).
4. Discussion
The results of our study demonstrate that the popu-lations of cultivable rhizobacteria were composed of three distinct groups. These three groups seem to be dominated by distinct taxa: the genus Pseudomonas
(Group A), the genus Cytophaga(Group B) and the Gram positives (Group C).
Bacteria from the genusPseudomonasare known to be the main colonisers of the rhizosphere. They are motile, ¯agellated rods that can metabolise a wide range of substances and are, thus, adapted to exploit-ing the diverse compounds in the root exudates (Sta-nier et al., 1980; Curl and Truelove, 1986). Bacteria of the genus Cytophaga, show gliding motility and can degrade various types of macromolecules such as cellulose, starch and protein (Christensen, 1977). They are well suited for decomposing the more com-plex substances in rhizodeposition.
Table 2
Fatty acid composition of bacteria in the three main groups. (For a precise definition of the groups, see text.)
Fatty acid Group A Group B Group C
16 : 0 29.3 2.5 3.2
Numbers of rhizobacterial isolates in the three main bacterial groups and in the dominating bacterial genera. (For a precise definition of the groups, see text.)
Genusa Group A Group B Group C
Pseudomonas 366 0 0
Unidentified isolates 297 198 16
The results indicating that Groups A and B were dominated by the genera Pseudomonas and Cyto-phaga, respectively, should be interpreted with cau-tion since in culturing the isolates we deviated slightly from the standard growth conditions for MIS. The incubation temperature in our analysis was 248C instead of 288C, and the concentration of the tryptic soybroth was 20 g/l instead of 30 g. These modi®ca-tions were, however, considered necessary since in our preliminary tests there were many isolates that showed poor growth under the speci®ed standard conditions. Haack et al. (1994), who investigated the effects of incubation temperature (17 and 328C) and growth medium composition, concluded that these factors did in¯uence the fatty acid composition of their bacterial strains but that the characteristic pro®les
of the genera generally were unaffected. Our results are in good agreement with their conclusion. Thus, although the deviations from the recommended growth conditions in our study decreased the relia-bility with which we were able to determine the isolates to the species level based on the MIS system, we were still able to reliably classify them to genus level.
Furthermore, not all isolates were identi®ed with a suf®cient degree of certainty. With the MIS, we were able to identify 57% of the bacteria (SI > 0.6). Thus, the remainder had to be listed as unidenti®ed. With such a great proportion of unidenti®ed bacteria in a population, nothing could be concluded about the population as a whole. Hence, we used the fatty acid composition to group the isolates. This way, all
iso-Table 4
The fatty acid composition of bacteria in subgroups of the three main groups. (For a precise definition of the main groups, see text.)
Fatty acid Group A Group B Group C
Pseudomonasa Not Pseudo.a
Cytophaga Not Cytophaga
Arthrobacter Bacillus Not Arthrobacter or Bacillus
16 : 0 31.7 26.9 2.6 2.4 2.5 2.5 3.7
16 : 1/15 : 0 Iso 2OH 31.5 29.2 11.9 11.8 1.1 0.0 2.8
18 : 1 14.1 10.3 0.0 0.0 0.0 0.0 0.2
12 : 0 2.8 4.2 0.0 0.0 0.0 0.0 0.4
17 : 0 CYCLO 5.9 5.4 0.0 0.0 0.0 0.0 0.0
10 : 0 3OH 3.4 4.4 0.0 0.0 0.0 0.0 0.1
12 : 0 3OH 4.0 4.5 0.0 0.1 0.0 0.0 0.1
12 : 0 2OH 4.5 2.6 0.0 0.0 0.0 0.0 0.0
12 : 1 3OH 0.2 3.4 0.0 0.0 0.0 0.0 0.0
Sum 98.0 90.8 14.6 14.4 3.6 2.5 7.3
SD of sum 1.0 14.2 5.8 8.7 2.5 1.4 10.8
15 : 0 Iso 0.0 0.2 28.4 30.7 12.9 25.0 11.4
17 : 1 Iso 0.0 0.0 9.3 6.1 0.0 0.0 0.1
17 : 0 Iso 3OH 0.0 0.0 11.6 10.3 0.0 0.0 0.0
15 : 0 0.1 0.3 4.8 4.1 0.3 0.2 0.3
15 : 1 0.0 0.0 2.7 2.2 0.0 0.0 0.0
17 : 1 0.0 0.0 2.0 1.6 0.0 0.0 0.0
17 : 1 Iso/Anteiso 0.0 0.0 2.0 1.5 0.1 0.5 0.0
15 : 1 Iso G 0.0 0.0 1.9 1.4 0.0 0.0 0.0
16 : 0 Iso 3OH 0.0 0.0 1.8 1.4 0.0 0.0 0.0
15 : 0 Iso 3OH 0.0 0.0 7.8 6.1 0.0 0.0 0.0
Sum 0.1 0.7 72.3 70.2 13.5 25.7 11.9
SD of sum 0.2 3.0 8.4 13.3 7.2 13.7 5.6
15 : 0 Anteiso 0.0 0.2 3.2 4.3 59.8 52.3 57.1
SD of 15 : 0 Ant. 0.1 1.9 0.8 4.3 9.9 13.4 13.3
lates could be characterised by their fatty acid pro®le irrespective of their assignment to a particular genus. Two dominating and distinct groups of bacteria, A and B, were thus recognised using the statistical analysis described above. Within each of these two groups, a certain proportion of isolates was identi®ed with a suf®ciently high degree of certainty. Let us assume that the identi®ed and unidenti®ed bacteria that cluster together when comparing their fatty acid pro®les also have other important properties in com-mon. If this assumption is valid, then our results indicate that the rhizobacterial populations are com-posed of two distinct and dominating groups that differ from each other in terms of motility type and nutri-tional requirements. These groups would occupy dif-ferent ecological niches determined by the composition of the rhizodeposition.
On average over the investigated soils and treat-ments, bacteria belonging to Group B accounted for approximately 37% of the population. If all of them belonged to theCytophagaorFlavobacteriumgenera, then the relative abundance of these genera was unusually high compared with other investigations (Kleeberger et al., 1983; Lambert et al., 1990; Kremer et al., 1990; Buyer and Kaufman, 1996). To our knowledge,Cytophaga/Flavobacteriumisolates have only been reported to constitute a high portion of the sampled rhizobacteria in one paper (Sato and Jiang, 1996b). It is crucial to determine the degree to which the composition of the sampled population re¯ects the composition of the original population of viable rhi-zobacteria. Incubation temperature and isolation med-ium are important factors that affect the viability of bacteria and, thus, the size of the recorded popula-tions. In the above-mentioned investigations the incu-bation temperature was between 22 and 308C, whereas we used 118C. Our lower temperature more closely resembles soil temperatures in temperate areas and
thus should better re¯ect the conditions to which the original population was exposed.
Acknowledgements
We thank A. Gidlund and L. FaÈreby for excellent technical assistance. Financial support was provided by the Swedish Council for Forestry and Agricultural Research.
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